Metabolic Switch and Cytotoxic Effect of Metformin on Burkitt Lymphoma

Altered cellular energetic metabolism has recently emerged as important feature of neoplastic cells. Indeed, interfering with cancer cell metabolism might represent a suitable therapeutic strategy. In this study, we aimed to assess glucose metabolism activation in human lymphomas and evaluate how metformin can exert its action on lymphoma cells. We studied a large series of human lymphomas (N = 252) and an in vitro model of Burkitt lymphoma (BL) cells. We combined molecular biology techniques, including global gene expression profiling (GEP) analysis, quantitative PCR (qPCR) and Western blotting, and biochemical assays, aimed to assess pentose phosphate pathway, tricarboxylic acid (TCA) cycle, and aerobic glycolysis rates. We found that glucose metabolism is overall enhanced in most lymphoma subtypes, based on gene expression profiling (GEP), with general shift to aerobic glycolysis. By contrast, normal B cells only showed an overall increase in glucose usage during germinal center transition. Interestingly, not only highly proliferating aggressive lymphomas but also indolent ones, like marginal zone lymphomas, showed the phenomenon. Consistently, genes involved in glycolysis were confirmed to be overexpressed in BL cells by qPCR. Biochemical assays showed that while aerobic glycolysis is increased, TCA cycle is reduced. Finally, we showed that metformin can induce cell death in BL cells by stressing cellular metabolism through the induction of GLUT1, PKM2, and LDHA. In conclusion, we unveiled glucose metabolism abnormalities in human lymphomas and characterized the mechanism of action of metformin in Burkitt lymphoma model.

Peripheral blood lymphocytes (PBL) obtained from three normal healthy blood donors were used as controls. Briefly, white blood cells were separated from red blood cells and granulocytes by separation of blood samples onto Ficoll-Paque density gradient media (Sigma, St. Louis, MO, USA). Next, monocytes were separated from lymphocytes by adhesion of suspension cells to petri dishes. The supernatant, containing the cells of interest, was then collected, cultured in completed RPMI-1640 and treated as already described for DAUDI cells.

Pentose phosphate pathway, TCA cycle and aerobic glycolysis rate measurement
DAUDI and PBL cells treated or non-treated with metformin were subjected to rate determination of pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) rates. In case of PPP, it was determined by incubating cells with tracer doses of [1-C 14 ]-glucose and [6-C 14 4]-glucose. After its internalization, glucose is rapidly phosphorylated to glucose 6-phosphate (G6P) to enter glycolysis. G6P can also be shunted to PPP. In the oxidative branch of PPP, it is first oxidized to 6-phosphogluconate and then decarboxylated to ribulose 5-phosphate with the consequent emission of CO 2 . Emission of CO 2 can be monitored by administration of [1-C 14 ]glucose. G6P, which continues with glycolysis, can enter the tricarboxylic acid cycle (TCA) encountering two subsequent decarboxylations. Emissions of CO 2 during TCA can be monitored by administration of [6-C 14 ]-glucose. The PPP rate was calculated as the difference between the 14 CO 2 derived from [1-C 14 ]-glucose (metabolized in both PPP and TCA) and that derived from [6-C 14 ]-glucose (metabolized only in the TCA). To this end, cells were cultured in completed RPMI containing traces of either [1-C 14 ]-glucose or [6-C 14 ]-glucose. Trapping of the radioactive 14 CO 2 metabolized from cells and scintillation counting was performed as previously reported 13 .
Aerobic glycolysis rate was calculated based on lactate production. DAUDI or PBL cells treated or non-treated with metformin were collected, counted for normalization and subjected to lactate analysis using Lactate Assay Kit (Sigma, Cat.# MAK064) according to the manufacturer's instructions. Results are expressed as micromoles of glucose consumed in the aerobic pathway to produce lactate.

Gene expression analysis by RT-qPCR
After 24-hours of treatment with 10 mM metformin, total RNA was extracted from DAUDI cells and PBL (as control) using RNeasy mini kit (Qiagen, Hilden, Germany). The respective cDNA was obtained by SuperScript VILO cDNA Synthesis kit (Invitrogen, UK) according to the manufacturer's instructions. Reverse transcription quantitative PCR (RT-qPCR) was then performed to evaluate the mRNA expression levels of six genes involved in the various branches of glucose metabolism: glucose transporter 1 (GLUT-1), hexokinase-1 (HK1), hexokinase-2 (HK2), pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA) and voltage-dependent anion channel 1 (VDAC1). Beta-actin (ACTB) was used as housekeeping gene for normalization. RT-qPCR reactions were carried out by using TaqMan Gene Expression Assay and TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 7500 Real-Time PCR System. The 20 μl PCR mixture consisted of 30 ng cDNA, 10 μl 2X TaqMan Gene Expression Master Mix, 1 μl of 20X TaqMan Gene Expression Assay and 5 μl of RNase and DNase-free deionized water. The reaction mixtures were incubated at 50°C for 2min, followed by 95 °C for 15 min and by 40 amplification cycles at 95 °C for 15 s, 60 °C for 1 min, as manufacturer instructions. The relative mRNA expression of each candidate gene was expressed as the ΔCt = Ct(target gene) -Ct(reference gene). In a further step, a second relative parameter was added as produced by the 1/2^ΔCt method. Statistical analyses were performed on the ΔCt values.

Western Blotting
Western blots were performed on DAUDI cells and primary lymphocytes after 24h treatment with metformin at a concentration of 10 mM. Protein extracts were obtained by cell lysis in RIPA buffer after suitable washes in PBS. Protein samples were separated onto SDS-PAGE and transferred to nitrocellulose membrane for immunoblotting. Monoclonal antibodies (mAb) used for the analyses were anti-LDHA (Cell Signalling Technologies, cat. #2012), anti-PKM (cat.#3198) and anti-GLUT-1 rabbit (cat.#12939). Beta-actin mouse mAb (cat. #3700) was used as a loading control. Goat Anti-Rabbit IgG-HRP Conjugate and Goat Anti-Mouse IgG-HRP Conjugate (BioRad, cat. #1706515 and cat.#1706516, respectively) were used as secondary antibodies. Primary antibodies were used at 1:1000 dilution, while secondary antibodies were used at 1:2000 dilution, according to the manufacturer's' instructions. The chemiluminescence reaction was performed by the WesternBright ECL HRP substrate kit (Advansta) and visualized by ChemiDoc MP Imaging system (BioRad). Band intensities were finally calculated by using Image Lab Software (BioRad  In the matrix, each column represents a sample and each row represents a gene. The color scale bar shows the relative gene expression changes normalized by the standard deviation (0 is the mean expression level of a given gene).